Activation of an α2A-adrenoceptor–Gαo1 fusion protein dynamically regulates the palmitoylation status of the G protein but not of the receptor

Barclay, Elaine; O’Reilly, Mark; Milligan, Graeme

doi:10.1042/bj20041432

Cited by 15 publications

(13 citation statements)

References 46 publications

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“…The defined 1:1 stoichiometry of the partner proteins is of particular use in measures of agonist-induced GTPase turnover number (Moon et al, 2001) and the regulation [coordinated (Stevens et al, 2001) or otherwise (Barclay et al, 2005)] of posttranslational thioacylation of GPCR and G protein and the effects of mutations in either partner that alter protein steady-state expression levels (Ward and Milligan, 2002). In the current studies, we have generated and explored the function and pharmacology of fusions between each of the DOP, KOP, and MOP opioid receptors with G i1␣ .…”

Section: Discussionmentioning

confidence: 99%

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Pascal¹,

Milligan²

2005

Mol Pharmacol

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Complementation of function after coexpression of pairs of nonfunctional G protein-coupled receptors that contain distinct inactivating mutations supports the hypothesis that such receptors exist as dimers. Chimeras between members of the metabotropic glutamate receptor-like family have been particularly useful because the N-terminal ligand binding and heptahelical transmembrane elements can be considered distinct domains. To examine the utility of a related approach for opioid receptors, fusion proteins were generated in which a pertussis toxin-resistant (Cys Extensive literature now exists on the capacity of a wide range of G protein-coupled receptors (GPCRs) to form dimers and/or higher-order oligomers (Lee et al., 2003;Breitwieser, 2004;Milligan, 2004). Despite this, many of the reports have been predominantly descriptive and provide limited insights into the proportion of different GPCRs that may exist as dimers, the relative propensity of different GPCRs to oligomerize, the molecular basis of dimerization, and whether there are differences in the details of how closely related GPCRs form dimers/oligomers.The ability of the DOP, KOP, and MOP opioid receptor subtypes to form homodimers and/or higher-order oligomers has previously been investigated using both coimmunoprecipitation and resonance energy transfer techniques (Cvejic and Devi, 1997;George et al., 2000;McVey et al., 2001;Li-Wei et al., 2002;Ramsay et al., 2002). Despite this, little information is available on the issues noted above, although informatic analysis has suggested potential interfaces in transmembrane helices that may contribute to opioid receptor subtype homodimerization (Filizola and Weinstein, 2002).If coexpression of two nonequivalent and nonfunctional mutants of a GPCR is both able and required to reconstitute receptor ligand binding and/or function, this can provide evidence in favor of direct protein-protein interactions and quaternary structure for the active receptor (Milligan and Bouvier, 2005). For example, coexpression of two forms of the angiotensin AT1 receptor that were unable to bind angiotensin II or related ligands because of point mutations in transmembrane region III or V restored ligand binding (Monnot et al., 1996). Such an approach has also been used to explore mechanisms of dimerization. Theoretical models of GPCR dimerization include both "contact" and "domain swap" dimers. Using the histamine H1 receptor as a model, Bakker These studies were supported, in part, by a Scottish Enterprise "Proof of concept" award (to G.M.).Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

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Section: Discussionmentioning

confidence: 99%

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Pascal¹,

Milligan²

2005

Mol Pharmacol

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“…The addition of the agonist adrenaline dramatically reduced palmitoylation of Go ␣ 1 in a fusion protein with the ␣ 2 A-adrenoceptor. The reduction in palmitoylation is concentration-dependent and correlates with the occupancy of the ligandbinding site [53] . This suggests that palmitoylation is directly associated with agonist stimulation and may be involved in regulating signals.…”

Section: Lipid Modificationmentioning

confidence: 99%

Molecular Mechanisms of Go Signaling

2009

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Go is the most abundant G protein in the central nervous system, where it comprises about 1% of membrane protein in mammalian brains. It functions to couple cell surface receptors to intercellular effectors, which is a critical process for cells to receive, interpret and respond to extracellular signals. Go protein belongs to the pertussis toxin-sensitive Gi/Go subfamily of G proteins. A number of G-protein-coupled receptors transmit stimuli to intercellular effectors through Go. Go regulates several cellular effectors, including ion channels, enzymes, and even small GTPases to modulate cellular function. This review summarizes some of the advances in Go research and proposes areas to be further addressed in exploring the functional role of Go.

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“…[1][2][3][4][5][6][7][8][9] a 2 -Adrenoceptors can be grouped into three highly homologous subtypes (a 2A , a 2B , and a 2C ) and, because of the difference in pharmacology, [10] a fourth subtype (a 2D ) can be formally distinguished, though this is rather a species orthologue.…”

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Structure‐Based Calculation of Binding Affinities of α_2A‐Adrenoceptor Agonists

et al. 2007

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Dedicated to Professor E. Sylvester Vizi on the occasion of his 70th birthday.Adrenergic receptors of the a 2 type (a 2 -adrenoceptors) belong to the family of seven transmembrane-spanning G-proteinlinked receptors.[1-9] a 2 -Adrenoceptors can be grouped into three highly homologous subtypes (a 2A , a 2B , and a 2C ) and, because of the difference in pharmacology, [10] a fourth subtype (a 2D ) can be formally distinguished, though this is rather a species orthologue.In general, the a 2 -adrenoceptors are responsible for the presynaptic feedback of the release of adrenaline and noradrenaline, their physiological agonists. Although numerous findings are available on the receptor subtypes from experiments with knockout mice [11] and these results are of some relevance for human pharmacology, the similar patterns of expression of adrenergic receptors in human and mouse tissues do not guarantee similar functions. Thus, the individual roles of the three a 2 -adrenoceptor subtypes in humans have not been completely elucidated. However, the results of the reported studies do indicate (see Supporting Information) that the a 2 -adrenoceptor subtypes are involved in various important physiological processes, and further investigations of the differences in their molecular pharmacology are therefore essential.The identification of subtype-specific functions from pharmacological experiments is currently not possible because of the lack of subtype-specific ligands [3,[6][7][8] and the cross-reactivity with imidazoline receptors.[7] The development of subtype-selective agonists would be useful as it would facilitate further examinations of the molecular pharmacology of the a 2 -adrenoceptors. The rational, structure-based design of such agonists requires a precise knowledge of the molecular structure of the binding site. Unfortunately, because of the difficulties inherent in crystallization, atomic-resolution structures of the a 2 -adrenoceptors are not available in the Protein Databank.In the present study, an atomic-resolution model of the a 2A -adrenoceptor was constructed through use of its amino acid sequence and the crystallographic bovine rhodopsin structure as a template. Similar homology models were earlier constructed by other researchers [12] and successfully used to provide qualitative explanations. The a 2A -adrenoceptor model in the present study is based on a crystallographic template structure with a resolution of 2.2 [13] appropriate for quantitative investigations (for details, refer to the Computational Methods below).In possession of the atomic resolution target structure (a 2A -adrenoceptor), 15 known agonist ligands were automatically docked to the presumed binding region of the receptor (Figure 1 a). Inspection of the results revealed that the docked ligand conformations are in physical contact with the key residues D3.32A C H T U N G T R E N N U N G (113), S5.42A C H T U N G T R E N N U N G (200), and S5.46A C H T U N G T R E N N U N G (204), previously identified by site-directed mutagenesis studies. [1...

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Activation of an α2A-adrenoceptor–Gαo1 fusion protein dynamically regulates the palmitoylation status of the G protein but not of the receptor

Cited by 15 publications

References 46 publications

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Molecular Mechanisms of Go Signaling

Structure‐Based Calculation of Binding Affinities of α_2A‐Adrenoceptor Agonists

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Activation of an α2A-adrenoceptor–Gαo1 fusion protein dynamically regulates the palmitoylation status of the G protein but not of the receptor

Cited by 15 publications

References 46 publications

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Molecular Mechanisms of Go Signaling

Structure‐Based Calculation of Binding Affinities of α2A‐Adrenoceptor Agonists

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Product

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Structure‐Based Calculation of Binding Affinities of α_2A‐Adrenoceptor Agonists